RESUMO
A monoclonal antibody (MAb) A2 was produced against a major polypeptide of Mycoplasma gallisepticum with a molecular mass of 64 kDa. MAb A2 reacted in immunoblot at a titre of 10(4.33) and had a titre of 10(4.5) in an enzyme-linked immunosorbent assay. In a radioimmunoprecipitation assay (RIPA) using metabolic [35S]methionine radiolabelling of M. gallisepticum suspension in Vero cell culture, MAb A2 was able to precipitate the 64 kDa protein and another protein of 47 kDa. The present study involving [35S]methionine labelling of M. gallisepticum in Vero cells represents a novel approach for labelling and characterizing the conformation-dependent mycoplasmal antigens in a RIPA system.
Assuntos
Antígenos de Bactérias/imunologia , Mycoplasma/imunologia , Ensaio de Radioimunoprecipitação/métodos , Animais , Chlorocebus aethiops , Marcação por Isótopo , Metionina , Radioisótopos de Enxofre , Células VeroRESUMO
A simple and improved procedure for radioimmunoprecipitation (RIPA) for the identification of major immunogenic proteins of avian reovirus using murine monoclonal and chicken polyclonal antibodies is described. Bacterial proteins (Staphylococcus aureus-Protein A or Streptococcus species-Protein G) commonly used in RIPA procedures lack reactivities with low avidity monoclonals belonging to immunoglobulin (Ig)G or IgM subtypes, as well as avian Ig. Hence we used an indirect approach utilizing species-specific anti-IgM, IgG or chicken Ig antibodies followed by the precipitation of the immune complexes using Protein A. This improved the sensitivity of the RIPA enabling the antigenic analysis of the major antigenic proteins of avian reovirus. The results indicated that the virus is highly immunogenic in the natural host, chicken than in mice. Furthermore, there exists a direct correlation between a strong neutralizing antibody response and an increased precipitation of the sigma (sigma) proteins of the virus. The results also demonstrate a strong association between the conformational viral epitopes of the three classes of proteins, large (lambda), medium (mu) and small (sigma).
Assuntos
Anticorpos Antivirais/imunologia , Testes de Precipitina/métodos , Radioimunoensaio/métodos , Reoviridae , Proteínas Virais/análise , Animais , Anticorpos Monoclonais/imunologia , Galinhas , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização/métodosRESUMO
Infectious bursal disease virus (IBDV) is a lymphotropic virus with cytocidal effect on B lymphocytes of the bursa of Fabricius. We investigated the susceptibility of clonal populations of reticuloendotheliosis virus-transformed chicken B lymphocytes of both spleen and bursal origin to IBDV infection. The infected cells were metabolically-labelled and the viral polypeptides were analyzed by immunoprecipitation using monoclonal antibodies (MAbs). Virus adsorption and the effects of neutralizing convalescent antisera and MAbs on virus attachment were studied using flow cytometry. The results of the study indicate firstly that the transformed B cells support virus replication and provide an efficient system for studying IBDV-lymphocyte interactions. Secondly, results obtained also showed that the most potent neutralizing antibodies may not be those involved in preventing the receptor-mediated viral attachment but rather those involved in the inhibition of downstream events such as virus penetration or uncoating.
Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/microbiologia , Citometria de Fluxo , Vírus da Doença Infecciosa da Bursa/imunologia , Animais , Anticorpos Monoclonais , Bolsa de Fabricius , Linhagem Celular Transformada , Separação Celular , Células Cultivadas , Embrião de Galinha , Galinhas , Fibroblastos , Soros Imunes , Vírus da Doença Infecciosa da Bursa/fisiologia , Testes de Neutralização , Receptores Virais/metabolismo , Vírus da Reticuloendoteliose , Baço , Replicação ViralRESUMO
Monoclonal antibodies (MAbs) neutralizing only the infectious bursal disease virus strains (IBDV) belonging to serotype 1 also immunoprecipitated the heterologous major antigenic proteins of serotype 2 IBDV. Detergent-solubilization followed by radioimmunoprecipitation assays (RIPA) using the MAbs revealed structural similarities between the conformation-dependent antigenic determinants of IBDV of the two existing serotypes. The presence of non-ionic detergent Triton X-100 determined the binding of altered proteins by MAbs in RIPA.
Assuntos
Detergentes , Vírus da Doença Infecciosa da Bursa/classificação , Animais , Anticorpos Monoclonais , Epitopos/química , Ensaio de Radioimunoprecipitação , Sorotipagem , Solubilidade , Células VeroRESUMO
Genomic segments of 10 selected isolates of avian reoviruses recovered from the intestine of birds affected with malabsorption syndrome or runting/stunting syndrome were separated by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analysed, depending on the period of recovery and particular geographic locations. The analysis showed great variability in the dsRNA profiles of the isolates and higher mobility of the segments L1, S1, S2, S3 and S4. There was no correlation between electropherotype and geographic origin of the isolate. The analysis also showed the emergence of electropherotypically distinct strains since the introduction of modified live reovirus vaccines.
Assuntos
Galinhas , Síndromes de Malabsorção/veterinária , Doenças das Aves Domésticas/microbiologia , Infecções por Reoviridae/veterinária , Reoviridae/classificação , Animais , Eletroforese em Gel de Poliacrilamida , Síndromes de Malabsorção/epidemiologia , Síndromes de Malabsorção/microbiologia , Doenças das Aves Domésticas/epidemiologia , RNA de Cadeia Dupla/análise , RNA Viral/análise , Reoviridae/genética , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/microbiologia , Células VeroRESUMO
Eggs and blood were collected from 11 hens in each of nine broiler-breeder flocks in Quebec. Serum and egg-yolk extracts were assayed for antibody titers to infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and reovirus (RV) by a commercial enzyme-linked immunosorbent assay (ELISA) kit. Comparison was made between egg-yolk and serum antibody titers by a regression analysis. A high correlation was observed between serum and yolk antibody titers to all the viruses tested (r = 0.9 for IBDV, 0.84 for IBV, 0.84 for NDV, and 0.91 for RV). Antibody monitoring of commercial breeder flocks using egg yolk instead of serum with commercial ELISA plates is thus feasible and is recommended.
Assuntos
Anticorpos Antivirais/análise , Galinhas/imunologia , Gema de Ovo/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Animais , Galinhas/sangue , Feminino , Vírus da Doença Infecciosa da Bursa/imunologia , Vírus da Doença de Newcastle/imunologia , Análise de Regressão , Reoviridae/imunologiaRESUMO
Turkeys poults were inoculated intraperitoneally with hemorrhagic enteritis virus (HEV) at 4-1/2 weeks of age. Antibody response and sequential development of viral antigen in various tissues were monitored. An enzyme-linked immunosorbent assay (ELISA) was developed to study antibody production, and immunoperoxidase staining was used to determined sites of localization of the viral antigens in tissues. Results of ELISA and immunodiffusion tests were compared. ELISA detected antibody from day 3 post-infection (p.i.), and gel diffusion detected antibody from day 5 p.i. Peak ELISA antibody titer appeared from day 14 p.i. HEV antigen was detected from 2-6 days p.i. in the spleen, liver, intestine, kidney, and bone marrow; peak titers in the spleen were on day 3 p.i. Virus was not detected after day 6 p.i.
Assuntos
Infecções por Adenoviridae/veterinária , Adenoviridae/imunologia , Anticorpos Antivirais/biossíntese , Aviadenovirus/imunologia , Enterite/veterinária , Doenças das Aves Domésticas/imunologia , Perus , Infecções por Adenoviridae/imunologia , Animais , Antígenos Virais/isolamento & purificação , Enterite/imunologia , Ensaio de Imunoadsorção Enzimática , Hemorragia Gastrointestinal/imunologia , Hemorragia Gastrointestinal/veterinária , Imunodifusão/veterinária , Técnicas ImunoenzimáticasRESUMO
Total RNA of eight avian reovirus isolates and the S1133 strain were compared by RNase T1-oligonucleotide mapping. The viruses were propagated in Vero cell cultures, and viral genomes were extracted from purified virions for comparison. Pairwise comparisons of the oligonucleotide maps showed genetic variation among reovirus isolates ranging from 78% to 99%. The T1 fingerprints of the RNA of isolates 1103, 724, 615, and 684 differed slightly from the standard S1133 strain, suggesting that the vaccine strain might have changed and became part of the circulating reoviruses. In contrast, when compared with the vaccine strain, isolates 902, 644, and 6207 showed greater differences in the fingerprint pattern. This genomic diversity may be due to the differences in immunological status of the affected avian population and/or due to simultaneous coinfection with different reovirus strains.
Assuntos
Oligonucleotídeos/análise , RNA Viral/análise , Reoviridae/genética , Vacinas Virais/genética , Animais , Reoviridae/classificação , Reprodutibilidade dos Testes , Vacinas Atenuadas/genética , Células VeroRESUMO
Infectious bursal disease viruses (IBDVs) were isolated from turkeys showing symptoms of arthritis and respiratory disease in commercial poultry farms in the province of Quebec, Canada. Synovial fluids collected from hock joints of arthritic birds and peripheral blood leukocytes obtained from the birds with respiratory problems were used for virus isolation in embryonated chicken eggs, and Vero and BGM-70 cell cultures. The infected cells were evaluated for the presence of IBDV by indirect immunofluorescence assay using monoclonal antibodies. The viruses were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral genome and by electron microscopy. Although one of these turkey isolates tested was neutralized by serotype 1-specific commercial chicken antisera, preliminary results indicated that there are antigenic differences between the Quebec isolate, IBDV QT-1, and the existing strains of IBDV belonging to serotype 1.
Assuntos
Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Doenças das Aves Domésticas/microbiologia , Infecções por Reoviridae/veterinária , Perus/microbiologia , Animais , Artrite Infecciosa/microbiologia , Artrite Infecciosa/veterinária , Linhagem Celular , Feminino , Imunofluorescência , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/ultraestrutura , Microscopia Eletrônica , Testes de Neutralização , Quebeque , RNA de Cadeia Dupla/análise , RNA Viral/análise , Infecções por Reoviridae/microbiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/veterinária , Líquido Sinovial/microbiologiaRESUMO
Chickens were experimentally infected with hemorrhagic enteritis virus, causing lesions similar to those observed in turkeys. Lesions were induced by intraperitoneal and oral infection. Virus particles with the morphology of adenovirus were demonstrated in the spleens of infected chickens. Splenic extracts from infected chickens caused lesions of hemorrhagic enteritis in turkeys. Recovery from the infection in chickens was followed by the production of antibodies to HEV, demonstrated by agar-gel precipitin test.
Assuntos
Infecções por Adenoviridae/veterinária , Galinhas , Doenças das Aves Domésticas/patologia , Infecções por Adenoviridae/patologia , Animais , Anticorpos Antivirais/análise , Testes de Precipitina , Baço/patologia , PerusRESUMO
A simple, rapid technique was developed for preparing chicken-embryo-skin cell cultures utilizing trypsinization of the skin of intact 12-day-old chicken embryos. When cell cultures were inoculated with fowl pox virus, those that consisted of at least 80% epithelial cells yielded a higher virus titer than fibroblast cell cultures.
Assuntos
Técnicas de Cultura/métodos , Pele , Animais , Divisão Celular , Células Cultivadas , Embrião de Galinha , Efeito Citopatogênico Viral , Células Epiteliais , Vírus da Varíola das Aves Domésticas/crescimento & desenvolvimento , Tripsina/metabolismo , Cultura de VírusRESUMO
In the spring of 1997, three large white hybrid turkey layer flocks of 52 wk of age experienced a severe respiratory condition. During the outbreak, the turkeys showed respiratory signs, an increased mortality rate, and an important drop in egg production. Macroscopic and histopathologic examinations were carried out on several carcasses, as well as bacteriologic analyses on different tissues. Colonies of Ornithobacterium rhinotracheale (OR) were detected after 24 hr of incubation, and the isolate appeared to be serotype A. The identification of the species was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. Since 1993, several cases of OR infection have been diagnosed in the United States and more recently in Canada. Monitoring of this emerging infection is recommended.
Assuntos
Surtos de Doenças/veterinária , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/veterinária , Doenças das Aves Domésticas/epidemiologia , Animais , Feminino , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/mortalidade , Pulmão/patologia , Necrose , Oviposição , Doenças das Aves Domésticas/mortalidade , Doenças das Aves Domésticas/patologia , Quebeque/epidemiologia , PerusRESUMO
Serum samples were collected from 1,075 clinically normal sheep and goats from 77 flocks in 7 agricultural regions of Quebec from June to August 1982. Sheep and goats were tested for antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, and bovine herpes-virus-1 by the indirect fluorescent antibody technique and for parainfluenza-3 virus by the hemagglutination inhibition test. The prevalence of antibodies in animals to respiratory syncytial virus was 31%; to bovine viral diarrhea virus, 22.2%; to bovine herpesvirus-1, 10.8%; and to parainfluenza-3 virus, 23.2%. Antibodies prevailed in similar proportions in young (less than 1 year) and adult (greater than 1 year) animals.
Assuntos
Anticorpos Antivirais/análise , Vírus da Diarreia Viral Bovina/imunologia , Cabras/imunologia , Herpesvirus Bovino 1/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Pestivirus/imunologia , Vírus Sinciciais Respiratórios/imunologia , Respirovirus/imunologia , Ovinos/imunologia , Animais , Imunofluorescência , QuebequeRESUMO
Interferon, fluorescent antibody, and neutralizing antibody responses were studied in sera of 9 calves inoculated with bovine respiratory syncytial virus, in relation to viral shedding and clinical signs of disease. The calves (5.5 to 6.5 weeks of age) were assigned to 3 groups. Group I was inoculated once with the virus, and groups II and III were challenge exposed at postinoculation day (PID) 15 or 37. Serum-neutralizing and indirect fluorescent antibody techniques were used to measure antibody responses. The plaque-inhibition technique, using vesicular stomatitis virus, was applied to measure serum interferon titers. The virus was recovered by inoculation of nasal secretions onto cell cultures. Fluorescent antibody was detected in all calves on PID 3, with maximum titers appearing approximately on PID 10. Low neutralizing antibody was detected in most animals on PID 3, and titers peaked approximately 4.5 weeks after inoculation and then decreased. Interferon titers were high in all calves during the early stage of infection, dropped to undetectable amounts by PID 6, and reappeared in low amounts at least 1 week later. All infected calves manifested clinical signs of disease by PID 4 to 9. Clinical signs of disease were not observed after challenge exposure at PID 15 or 37, and anamnestic responses were not detected. Virus was recovered after challenge exposure at PID 15, but not at PID 37.
Assuntos
Anticorpos Antivirais/análise , Doenças dos Bovinos/imunologia , Interferons/sangue , Vírus Sinciciais Respiratórios/imunologia , Infecções por Respirovirus/veterinária , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Imunofluorescência/veterinária , Testes de Neutralização/veterinária , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/microbiologiaRESUMO
A herd of 125 Holstein cows manifested fertility problems for two years. The number of services per pregnancy was 2.97, conception rate was 33% after the first service, and the average number of open days was 127. Abortions occurred in four cows over the last 12 months. The herd was not vaccinated against any disease. Natural service by a bull and artificial insemination were used for breeding the cows. Bovine herpesvirus type 1 was demonstrated in sperm heads from the bull by direct and indirect fluorescent antibody techniques, and the virus was isolated on cell cultures. The virus was also isolated from the uterine secretions of some cows and from two aborted fetuses.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Herpesviridae/veterinária , Herpesviridae/isolamento & purificação , Infertilidade Feminina/veterinária , Espermatozoides/microbiologia , Aborto Animal/etiologia , Animais , Bovinos , Feminino , Infecções por Herpesviridae/microbiologia , Infertilidade Feminina/etiologia , Masculino , GravidezAssuntos
Aborto Animal/microbiologia , Monócitos/microbiologia , Vírus de RNA/crescimento & desenvolvimento , Transtornos Respiratórios/veterinária , Doenças dos Suínos/microbiologia , Viroses/veterinária , Animais , Células Cultivadas , Efeito Citopatogênico Viral , Feminino , Gravidez , Vírus de RNA/imunologia , Transtornos Respiratórios/microbiologia , Suínos , Síndrome , Viroses/microbiologiaRESUMO
Monoclonal antibodies (MAbs) to a local turkey isolate (QT-1) of infectious bursal disease virus (IBDV) were produced to identify the virus-specific neutralizing proteins. Radioimmunoprecipitation assays showed that all the MAbs were specific for major viral protein, VP2. Two of the MAbs neutralized the local turkey and chicken isolates along with a reference strain belonging to serotype 1 but not the reference strain of serotype 2. The reactivities of the neutralizing MAbs against two reference strains and some recent field strains of IBDV isolated in the province of Québec were studied by indirect enzyme-linked immunosorbent assay and virus neutralization tests. The variations in the reactivities of the MAbs observed suggest differences in the neutralizing epitopes of the different isolates. Competitive binding assay using the MAbs revealed the presence of a third epitope involved in the neutralization of IBDV belonging to serotype 1.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Doença Infecciosa da Bursa/imunologia , Testes de Neutralização , Animais , Anticorpos Monoclonais/imunologia , Capsídeo/imunologia , Proteínas do Capsídeo , Galinhas/microbiologia , Ensaio de Imunoadsorção Enzimática , Vírus da Doença Infecciosa da Bursa/classificação , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Ensaio de Radioimunoprecipitação , Sorotipagem , Perus/microbiologiaRESUMO
Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 microL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea.
Assuntos
Vírus da Diarreia Viral Bovina/isolamento & purificação , Herpesvirus Bovino 1/isolamento & purificação , Nariz/microbiologia , Pestivirus/isolamento & purificação , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Bovinos , Epitélio/microbiologia , Imunofluorescência , Imunização/veterinária , Rinotraqueíte Infecciosa Bovina/diagnóstico , Cultura de Vírus/métodosRESUMO
Reoviruses were isolated from intestinal contents of broiler chickens from nine flocks in Quebec with malabsorption syndrome. Serum neutralization test demonstrated the existence of antigenic differences between the isolates and the reference vaccine strain. The isolated reoviruses were inoculated orally and into the foot pad in one-day-old chicks, resulting in a transient, but significant depression in body weight gains. Chickens infected with isolate 615, showed in addition to growth problems, clinical signs and tissue lesions similar to those observed in field cases. When isolate 615 was inoculated into SPF chicks at one day of age, intestinal absorption of D-xylose in infected chicks at 7 days post-infection was significantly lower (P <0.05) than for corresponding controls. This study suggests the implication of some reovirus isolates, such as 615 which was serologically distinct from the vaccine strain S1133, as infectious agents associated with pathological conditions other than viral arthritis.